Preclinical Toxicity Evaluation of a Potent Oncolytic Virus Oncolytic viruses (OV) represent a relatively new therapeutic paradigm that employs the ability of viruses to infect tumor cells, multiply in them, destroy them and spread to other tumor cells in successive rounds. Over the last decade, a first wave of oncolytic viruses have been tested in the clinical realm, including OVs based on herpes simplex virus Type 1 (HSV-1). These clinical trials have not showed evidence of doselimiting toxicities in humans when injected directly into malignant gliomas or into brain surrounding the glioma, but evidence for efficacy in these small scale trials has also been lacking, probably due to their high degree of attenuation. We have now developed and published preclinical efficacy results on a new, more potent oncolytic virus (rQNestin34.5) that employs the human nestin promoter-enhancer element to drive expression of the HSV1 gene, Y134.5, encoding the viral protein ICP34.5. This strategy allows the virus to replicate to high yields in cells that express nestin, while replication in cells that do not express nestin is severely attenuated. We and others have shown that nestin is expressed in a large proportion of gliomas, while it is not expressed in normal adult brain. In fact, nestin is one of the markers reported to distinguish the glioma stem-like or initiator subpopulation thought to provide the self-renewal capabilities of these tumors. In addition, we have published that immunomodulation with cyclophosphamide further enhances the potency and antiglioma efficacy of rQNestin34.5. Therefore, at this juncture, our published efficacy studies, revealing impressive and significant antiglioma properties of this OV, encourage us to pursue preclinical toxicity studies in mice models of HSV1 toxicity (and non-human primate models) using preclinical lots of OV. We thus propose the following four specific aim/milestones to justify two future clinical trials in humans: Milestone/Aim 1: Bioequivalency of rQNestin34.5v.2 and of reagents and standard operating protocol for future clinical grade production; Milestone/Aim 2: Validation of therapeutic efficacy and safety monitoring with rQNestin34.5v.2; Milestone/Aim 3: Validation of safety monitoring with rQNestin34.5v.2 in presence of CPA: Milestone/Aim 4: Final toxicology/biodistribution study with GMP-grade clinical lot . The successful pursuit of milestone 4 will permit us to approach regulatory agencies to start a phase I trial in patients with recurrent malignant glioma using rQNestin34.5v.2 + CPA immomunodulation and subsequently a phase I trial in newly diagnosed glioma using rQNestin34.5v.2 + CPA immomunodulation + standard therapy. RELEVANCE: Malignant glioma remains a daunting challenge with minimal progress in the last 40 years in extending patient survival and time to tumor progression. The project will permit the development of a promising biologic agent as a new treatment for this cancer and allow its testing in human clinical trials.